Human earwax consists of wet and dry types. Dry earwax is frequent in East Asians, whereas wet earwax is common in other populations. Here we show that a SNP, 538G --> A (rs17822931), in the ABCC11 gene is responsible for determination of earwax type. The AA genotype corresponds to dry earwax, and GA and GG to wet type. A 27-bp deletion in ABCC11 exon 29 was also found in a few individuals of Asian ancestry. A functional assay demonstrated that cells with allele A show a lower excretory activity for cGMP than those with allele G. The allele A frequency shows a north-south and east-west downward geographical gradient; worldwide, it is highest in Chinese and Koreans, and a common dry-type haplotype is retained among various ethnic populations. These suggest that the allele A arose in northeast Asia and thereafter spread through the world. The 538G --> A SNP is the first example of DNA polymorphism determining a visible genetic trait.

We previously determined that expression of human multidrug resistance protein (MRP) 8, a recently described member of the MRP family of ATP-binding cassette transporters, enhances cellular extrusion of cyclic nucleotides and confers resistance to nucleotide analogs (J Biol Chem 278:29509-29514, 2003). However, the in vitro transport characteristics of the pump have not been determined. In this study, the substrate selectivity and biochemical activity of MRP8 is investigated using membrane vesicles prepared from LLC-PK1 cells transfected with MRP8 expression vector. Expression of MRP8 is shown to stimulate the ATP-dependent uptake of a range of physiological and synthetic lipophilic anions, including the glutathione S-conjugates leukotriene C4 and dinitrophenyl S-glutathione, steroid sulfates such as dehydroepiandrosterone 3-sulfate (DHEAS) and estrone 3-sulfate, glucuronides such as estradiol 17-beta-D-glucuronide (E(2)17betaG), the monoanionic bile acids glycocholate and taurocholate, and methotrexate. In addition, MRP8 is competent in the in vitro transport of cAMP and cGMP, in accord with the results of our previously reported cellular studies. DHEAS, E(2)17betaG, and methotrexate were transported with K(m) and V(max) values of 13.0 +/- 0.8 microM and 34.9 +/- 9.5 pmol/mg/min, 62.9 +/- 12 microM and 62.0 +/- 5.2 pmol/mg/min, and 957 +/- 28 microM and 317 +/- 17 pmol/mg/min, respectively. Based upon the stimulatory action of DHEAS on uptake of E(2)17betaG, the attenuation of this effect at high DHEAS concentrations and the lack of reciprocal promotion of DHEAS uptake by E(2)17betaG, a model involving nonreciprocal constructive interactions between some transport substrates is invoked. These results suggest that MRP8 participates in physiological processes involving bile acids, conjugated steroids, and cyclic nucleotides and indicate that the pump has complex interactions with its substrates.

MRP8 (ABCC11) is a recently identified cDNA that has been assigned to the multidrug resistance-associated protein (MRP) family of ATP-binding cassette transporters, but its functional characteristics have not been determined. Here we examine the functional properties of the protein using transfected LLC-PK1 cells. It is shown that ectopic expression of MRP8 reduces basal intracellular levels of cAMP and cGMP and enhances cellular extrusion of cyclic nucleotides in the presence or absence of stimulation with forskolin or SIN-1A. Analysis of the sensitivity of MRP8-overexpressing cells revealed that they are resistant to a range of clinically relevant nucleotide analogs, including the anticancer fluoropyrimidines 5'-fluorouracil (approximately 3-fold), 5'-fluoro-2'-deoxyuridine (approximately 5-fold), and 5'-fluoro-5'-deoxyuridine (approximately 3-fold), the anti-human immunodeficiency virus agent 2',3'-dideoxycytidine (approximately 6-fold) and the anti-hepatitis B agent 9'-(2'-phosphonylmethoxynyl)adenine (PMEA) (approximately 5-fold). By contrast, increased resistance was not observed for several natural product chemotherapeutic agents. In accord with the notion that MRP8 functions as a drug efflux pump for nucleotide analogs, MRP8-transfected cells exhibited reduced accumulation and increased efflux of radiolabeled PMEA. In addition, it is shown by the use of in vitro transport assays that MRP8 is able to confer resistance to fluoropyrimidines by mediating the MgATP-dependent transport of 5'-fluoro-2'-deoxyuridine monophosphate, the cytotoxic intracellular metabolite of this class of agents, but not of 5'-fluorouracil or 5'-fluoro-2'-deoxyuridine. We conclude that MRP8 is an amphipathic anion transporter that is able to efflux cAMP and cGMP and to function as a resistance factor for commonly employed purine and pyrimidine nucleotide analogs.

We previously determined that expression of human multidrug resistance protein (MRP) 8, a recently described member of the MRP family of ATP-binding cassette transporters, enhances cellular extrusion of cyclic nucleotides and confers resistance to nucleotide analogs (J Biol Chem 278:29509-29514, 2003). However, the in vitro transport characteristics of the pump have not been determined. In this study, the substrate selectivity and biochemical activity of MRP8 is investigated using membrane vesicles prepared from LLC-PK1 cells transfected with MRP8 expression vector. Expression of MRP8 is shown to stimulate the ATP-dependent uptake of a range of physiological and synthetic lipophilic anions, including the glutathione S-conjugates leukotriene C4 and dinitrophenyl S-glutathione, steroid sulfates such as dehydroepiandrosterone 3-sulfate (DHEAS) and estrone 3-sulfate, glucuronides such as estradiol 17-beta-D-glucuronide (E(2)17betaG), the monoanionic bile acids glycocholate and taurocholate, and methotrexate. In addition, MRP8 is competent in the in vitro transport of cAMP and cGMP, in accord with the results of our previously reported cellular studies. DHEAS, E(2)17betaG, and methotrexate were transported with K(m) and V(max) values of 13.0 +/- 0.8 microM and 34.9 +/- 9.5 pmol/mg/min, 62.9 +/- 12 microM and 62.0 +/- 5.2 pmol/mg/min, and 957 +/- 28 microM and 317 +/- 17 pmol/mg/min, respectively. Based upon the stimulatory action of DHEAS on uptake of E(2)17betaG, the attenuation of this effect at high DHEAS concentrations and the lack of reciprocal promotion of DHEAS uptake by E(2)17betaG, a model involving nonreciprocal constructive interactions between some transport substrates is invoked. These results suggest that MRP8 participates in physiological processes involving bile acids, conjugated steroids, and cyclic nucleotides and indicate that the pump has complex interactions with its substrates.

MRP8 (ABCC11) is a recently identified cDNA that has been assigned to the multidrug resistance-associated protein (MRP) family of ATP-binding cassette transporters, but its functional characteristics have not been determined. Here we examine the functional properties of the protein using transfected LLC-PK1 cells. It is shown that ectopic expression of MRP8 reduces basal intracellular levels of cAMP and cGMP and enhances cellular extrusion of cyclic nucleotides in the presence or absence of stimulation with forskolin or SIN-1A. Analysis of the sensitivity of MRP8-overexpressing cells revealed that they are resistant to a range of clinically relevant nucleotide analogs, including the anticancer fluoropyrimidines 5'-fluorouracil (approximately 3-fold), 5'-fluoro-2'-deoxyuridine (approximately 5-fold), and 5'-fluoro-5'-deoxyuridine (approximately 3-fold), the anti-human immunodeficiency virus agent 2',3'-dideoxycytidine (approximately 6-fold) and the anti-hepatitis B agent 9'-(2'-phosphonylmethoxynyl)adenine (PMEA) (approximately 5-fold). By contrast, increased resistance was not observed for several natural product chemotherapeutic agents. In accord with the notion that MRP8 functions as a drug efflux pump for nucleotide analogs, MRP8-transfected cells exhibited reduced accumulation and increased efflux of radiolabeled PMEA. In addition, it is shown by the use of in vitro transport assays that MRP8 is able to confer resistance to fluoropyrimidines by mediating the MgATP-dependent transport of 5'-fluoro-2'-deoxyuridine monophosphate, the cytotoxic intracellular metabolite of this class of agents, but not of 5'-fluorouracil or 5'-fluoro-2'-deoxyuridine. We conclude that MRP8 is an amphipathic anion transporter that is able to efflux cAMP and cGMP and to function as a resistance factor for commonly employed purine and pyrimidine nucleotide analogs.

The directed movement of organic anions into, out of or within a cell, or between cells, by means of some agent such as a transporter or pore. Organic anions are atoms or small molecules with a negative charge which contain carbon in covalent linkage.

Dehydroepiandrosterone 3-sulfate and other neurosteroids are synthesized in the CNS and peripheral nervous system where they may modulate neuronal excitability by interacting with ligand-gated ion channels. For this modulatory activity, neurosteroids have to be locally released from either neurons or glial cells. We here identify the integral membrane protein ABCC11 (multidrug resistance protein 8) as an ATP-dependent efflux pump for steroid sulfates, including dehydroepiandrosterone 3-sulfate, and localize it to axons of the human CNS and peripheral nervous system. ABCC11 mRNA was detected in human brain by real-time polymerase chain reaction. Antibodies raised against ABCC11 served to detect the protein in brain by immunoblotting and immunofluorescence microscopy. ABCC11 was preferentially found in the white matter of the brain and co-localized with neurofilaments indicating that it is an axonal protein. Additionally, ABCC11 was localized to axons of the peripheral nervous system. For functional studies, ABCC11 was expressed in polarized Madin-Darby canine kidney cells where it was sorted to the apical membrane. This apical sorting is in accordance with the localization of ABCC11 to the axonal membrane of neurons. Inside-out plasma membrane vesicles containing recombinant ABCC11 mediated ATP-dependent transport of dehydroepiandrosterone 3-sulfate with a Km value of 21 microM. This transport function together with the localization of the ABCC11 protein in vicinity to GABAA receptors is consistent with a role of ABCC11 in dehydroepiandrosterone 3-sulfate release from neurons to sites of dehydroepiandrosterone 3-sulfate-mediated receptor modulation. Our findings may provide a basis for the characterization of mutations in the human ABCC11 gene and their linkage with neurological disorders.

We previously determined that expression of human multidrug resistance protein (MRP) 8, a recently described member of the MRP family of ATP-binding cassette transporters, enhances cellular extrusion of cyclic nucleotides and confers resistance to nucleotide analogs (J Biol Chem 278:29509-29514, 2003). However, the in vitro transport characteristics of the pump have not been determined. In this study, the substrate selectivity and biochemical activity of MRP8 is investigated using membrane vesicles prepared from LLC-PK1 cells transfected with MRP8 expression vector. Expression of MRP8 is shown to stimulate the ATP-dependent uptake of a range of physiological and synthetic lipophilic anions, including the glutathione S-conjugates leukotriene C4 and dinitrophenyl S-glutathione, steroid sulfates such as dehydroepiandrosterone 3-sulfate (DHEAS) and estrone 3-sulfate, glucuronides such as estradiol 17-beta-D-glucuronide (E(2)17betaG), the monoanionic bile acids glycocholate and taurocholate, and methotrexate. In addition, MRP8 is competent in the in vitro transport of cAMP and cGMP, in accord with the results of our previously reported cellular studies. DHEAS, E(2)17betaG, and methotrexate were transported with K(m) and V(max) values of 13.0 +/- 0.8 microM and 34.9 +/- 9.5 pmol/mg/min, 62.9 +/- 12 microM and 62.0 +/- 5.2 pmol/mg/min, and 957 +/- 28 microM and 317 +/- 17 pmol/mg/min, respectively. Based upon the stimulatory action of DHEAS on uptake of E(2)17betaG, the attenuation of this effect at high DHEAS concentrations and the lack of reciprocal promotion of DHEAS uptake by E(2)17betaG, a model involving nonreciprocal constructive interactions between some transport substrates is invoked. These results suggest that MRP8 participates in physiological processes involving bile acids, conjugated steroids, and cyclic nucleotides and indicate that the pump has complex interactions with its substrates.

MRP8 (ABCC11) is a recently identified cDNA that has been assigned to the multidrug resistance-associated protein (MRP) family of ATP-binding cassette transporters, but its functional characteristics have not been determined. Here we examine the functional properties of the protein using transfected LLC-PK1 cells. It is shown that ectopic expression of MRP8 reduces basal intracellular levels of cAMP and cGMP and enhances cellular extrusion of cyclic nucleotides in the presence or absence of stimulation with forskolin or SIN-1A. Analysis of the sensitivity of MRP8-overexpressing cells revealed that they are resistant to a range of clinically relevant nucleotide analogs, including the anticancer fluoropyrimidines 5'-fluorouracil (approximately 3-fold), 5'-fluoro-2'-deoxyuridine (approximately 5-fold), and 5'-fluoro-5'-deoxyuridine (approximately 3-fold), the anti-human immunodeficiency virus agent 2',3'-dideoxycytidine (approximately 6-fold) and the anti-hepatitis B agent 9'-(2'-phosphonylmethoxynyl)adenine (PMEA) (approximately 5-fold). By contrast, increased resistance was not observed for several natural product chemotherapeutic agents. In accord with the notion that MRP8 functions as a drug efflux pump for nucleotide analogs, MRP8-transfected cells exhibited reduced accumulation and increased efflux of radiolabeled PMEA. In addition, it is shown by the use of in vitro transport assays that MRP8 is able to confer resistance to fluoropyrimidines by mediating the MgATP-dependent transport of 5'-fluoro-2'-deoxyuridine monophosphate, the cytotoxic intracellular metabolite of this class of agents, but not of 5'-fluorouracil or 5'-fluoro-2'-deoxyuridine. We conclude that MRP8 is an amphipathic anion transporter that is able to efflux cAMP and cGMP and to function as a resistance factor for commonly employed purine and pyrimidine nucleotide analogs.

Protein which is part of a reference proteome. Reference proteomes are a subset of proteomes that have been selected either manually or algorithmically according to a number of criteria to provide a broad coverage of the tree of life and a representative cross-section of the taxonomic diversity found within UniProtKB, as well as the proteomes of well-studied model organisms and other species of interest for biomedical research.